PROJECT SUMMARY Francisella tularensis is the causative agent of the zoonotic disease tularemia. F. tularensis is a highly virulent intracellular pathogen that is easily transmitted to humans when aerosolized and thus has the potential for use as a bioterrorism agent. The molecular basis for the high infectivity and virulence of F. tularensis is not well understood. As an intracellular pathogen, F. tularensis must evade cellular innate immune detection; however, a mechanistic understanding for how F. tularensis subverts the host response during infection is lacking. We have identified TolC as critical for the virulence of F. tularensis. TolC is an outer membrane channel protein involved in both multidrug efflux and the secretion of a wide range of bacterial effector proteins by the type I protein secretion pathway. We have evidence that factors secreted via TolC function to dampen innate immune responses of the host, including programmed cell death pathways, providing the bacteria extended time to replicate within the protected intracellular niche and prolonging survival within host tissues, thereby tipping the host-pathogen balance in favor of the pathogen. These host suppressive activities related to TolC function are likely key to the successful intracellular lifestyle of F. tularensis and critical to its extreme virulence. This proposal will investigate the mechanism of TolC in Francisella pathogenesis, with the overall goal of revealing molecular details by which intracellular pathogens interact with host cells, evade host defenses, and manipulate host responses. The first aim of the proposal will define the role of TolC in modulation of host responses during in vivo infection and the connection of these activities to F. tularensis virulence. The second aim of the proposal will identify Francisella effectors secreted via TolC, and by other routes, during host cell infection and will determine genes required for TolC-dependent subversion of host cell death pathways. The third aim of the proposal will determine the mechanism by which F. tularensis suppresses macrophage innate immune responses during infection in a TolC-dependent manner. Data generated by this proposal will lead to a detailed, mechanistic understanding of TolC function in F. tularensis. The identification of effectors or toxins secreted by F. tularensis would represent a significant advance for the field. This proposal will not only lead to better knowledge of F. tularensis virulence mechanisms, but will also provide new insights into strategies used by intracellular pathogens to survive within the host and cause disease.